Efficient neural differentiation of TW1 hESCs.

<p>(A) The Oct4 transcription factor, a well-characterized pluripotent marker, was detected in undifferentiated TW1 hESCs. (B) Dissociated hESCs aggregated in suspension and formed numerous embryonic bodies. (C, D) Differentiating hESCs exhibited reduced expression of Oct4 on D5 (C) and displayed a classical neural rosette conformation after plating on culture plates on D10 (D). (E–G) NPCs express N-cadherin (E), nestin (F), Pax6 and Sox1 transcription factors (G). (H) Extensive TuJ1⁺ neurites were detected in NPC-derived mature neurons on D25. Scale bars, 100 µm.</p

JEV infection in vimentin-expressing cells.

<p>(A, B) Vimentin expression was observed in most Sox1⁺ NPC cells and GFAP⁺ cells. (C, D) Most TuJ1⁺ expressing cells (matured neurons) did not express the vimentin. Panel C and D, low magnification and high magnification, respectively. (E, F, G) JEV-infected cells (JEV NS1⁺ cells, blue) were colocalized with the vimentin-expressing cells (green), but not the human mature neurons (TuJ1⁺, arrow head, red). Scar bar in each panel, 10 µm.</p

Viral replication and cytopathic effect of JEV-infected NPCs.

<p>(A) The viral growth curve in JEV-infected NPCs at MOI 0.1 is illustrated. (B, C) The infected NPCs expressed the viral NS1 and NS1′ proteins (B) and exhibited a depleted cell population (C) at MOI 0.1. (D) The degree of membrane externalization on the infected cells, detected by Annexin V staining, was quantified by flow cytometry analysis.</p

JEV infection in hESC-derived glial cells and neurons.

<p>(A) The JEV NS1 proteins were detected in GFAP-expressing glial cells at MOI 1 on 1 d.p.i.. (B–D) The expression of JEV NS1 proteins in mature neurons (TuJ1⁺) is illustrated at MOI 1 on 1 d.p.i. (B) and at MOI 10 on 1 d.p.i. (C) or 3 d.p.i. (D). (E–H) The infectivity of JEV was determined in GABAnergic (E), glutaminergic (F), dopaminergic (G) and serotonergic neurons (H) at MOI 10 on 3 d.p.i. TuJ1 antibody, anti-βIII tubulin. TH, tyrosine hydroxylase. Scale bar, 20 µm.</p

JEV infection in anti-vimentin pretreated NPCs.

<p>(A, B, C) Nestin⁺-NPCs (red) were pretreated with preimmune Abs (mock) (A), 1∶50 diluted (B) and 1∶100 diluted (C) anti-vimentin Abs for 1h at 4°C and then were infected with JEV at MOI 0.2 for 1h. The infected NPCs were fixed and stained at 24 h.p.i.. Scale bar, 50 µm. (D) The ratio of JEV-infected cells (expressing viral NS1 protein, green) were counted with the assistance of a densitometric software (total cells>5000, duplicated) (*, p<0.05; **, p<0.01, one-way ANOVA). (E) The released viral particles, collected from the supernatants of infected cells at 12 and 24 h.p.i., were titrated by plaque forming assay (*, p<0.05; **, p<0.01, one-way ANOVA). Representative data were collected from two independent experiments.</p